Hox genes are evolutionarily highly conserved, and regulate a network of genes controlling development of body plan. In particular, the role of Hox genes in formation of the appendages, namely the phallus and limbs, appear very similar. Regulation of Hox genes during appendage development involves lncRNAs. However, how HOX and adjacent lncRNAs regulate development of the phallus are largely unknown.
In marsupials, development of the phallus shows distinct differences in timing relative to the corresponding events in eutherian mammals. The neonatal phallus is identical from birth until after day 50 post partum (pp) when gene expression changes markedly and when male phallus growth accelerates. The morphological differentiation of the male phallus is modulated by androgen exposure between d 20-30 pp. In tammars, we can induce sex reversal of the urogenital organs, including the phallus. We therefore induced male-type phallus development in developing females by exposure to androgens (androstanediol) from day 25-30 and day 25-50 and induced hypospadias in developing males by castration at day 25 or by oestrogen treatment from birth to days 30 and 50. Phallic tissues were collected at day 30 and day 50 pp for RNA-Seq after these treatments. We focussed on the HOX genes and adjacent lncRNAs with RNA-Seq data and compared these to Chip-Seq data of human and mouse. There was more alternative splicing than expected, even for the highly conserved HOX genes. After analysis of all HOX genes and adjacent lncRNAs, we showed that while transcription levels of HOXD13 are not different between phallus tissues, the isoforms expressed were markedly different between male and females. Our findings were confirmed with specific qPCR and mRNA in situ hybridization. In conclusion, our data suggests that alternative splicing of key genes, such as HOX and their adjacent lncRNAs may regulate the development of the phallus.